The yield of a crop or ornamental plant ultimately depends on the energy the plant gains through the fixing of carbon dioxide (CO2) into carbohydrates during photosynthesis. The primary sources of photosynthesis are the leaves, and to a lesser extent stem tissue. Other organs of the plant, such as roots, seeds or tubers, do not make a material contribution to the formation of photoassimilates, and instead are dependent for their growth on the supply of carbohydrates received from photosynthetically active organs. This means there is a flow in photosynthetically gained energy from photosynthetically active tissues to photosynthetically inactive tissues.
The direction of phloem transport of this energy is determined by the relative locations of the areas of supply and utilization of the products of photosynthesis. Translocation occurs from areas of supply (sources) to areas of metabolism or storage (sinks). Sources include any exporting organ, typically a mature leaf that is capable of producing photosynthate in excess of its own needs. Another type of source is a storage organ during the exporting phase of its development. For example a storage root may be a sink during the first growing season when it accumulates sugars received from the source leaves. During the second growing season the same root could become a source, when the stored sugars are remobilized and utilized to produce a new shoot which ultimately becomes reproductive. Sinks include any non-photosynthetic organs of the plant and organs that do not produce enough photosynthetic products to support their own growth or storage needs. Roots, tubers, developing fruits and immature leaves which must import carbohydrate for normal development are all examples of sink tissues. Sink tissues differ in their ability to attract source products. Elements such as stress, developmental stages of plant tissues, and osmotic potential all affect the transport of photoassimilates.
Differential distribution of photoassimilates within the plant is termed partitioning. Partitioning of assimilated carbon amongst sink organs is a critical factor that controls rate and pattern of plant growth. The regulation of the diversion of fixed carbon into the various metabolic pathways is termed allocation. The rate of fixed carbon in a source cell can be classified into three principle categories; storage, utilization, and transport. Starch is synthesized and stored within chloroplasts and in most species is a primary storage form that is mobilized for translocation during the night. Fixed carbon can be utilized within various compartments of the photosynthesizing cell to meet energy needs of the cell or provide carbon skeletons for the synthesis of other compounds required by the cells. Fixed carbon can also be incorporated into transport sugars for export to various sink tissues.
The rate of photosynthesis of leaves is strongly influenced by the demands of the sink. There are cases in which senescent leaves can be rejuvenated to full photosynthetic performance when the sink/source ratio is increased substantially. On the other hand rapid growth of a sink can sometimes compete with leaves for remobilizable nitrogen leading to senescent of the leaf and a drop in its photosynthetic capacity. Young leaves normally act as a sink rather than as a source. After a certain time however they begin to export carbohydrates to the phloem although import carbohydrate may continue for a while through different vascular strands. Once sucrose begins to actively load into companion cells and then into the sieve elements, water will enter by osmosis and flow will begin out of the line of veins. The leaf will become a source instead of a sink.
Two primary photoassimilates are sugar and starch, and these products are important to yield and plant development. Sugar and starch biochemistry are interrelated in plants. (See, e.g., Sivak, M. N. and J. Preiss (1994). Starch synthesis in seeds. In: Seed development and germination. Kigel, J. and G. Galili, eds. (Marcel Dekker, New York), pp. 139-168; J. S. Hawker (1985). Sucrose. In: Biochemistry of storage carbohydrates in green plants. P. M. Dey and R. A. Dixon, Eds., (Academic Press, London), pp. 1-51, which are incorporated herein by reference).
During the early development of storage organs, such as seeds and tubers, sucrose is imported and used for building the cellular components required for growth and development. Following this phase the metabolic program changes to convert the imported sucrose into storage compounds such as starch in tubers and fatty acids in oil seeds. Metabolism is finally altered to convert the starch and oils into reduced carbon compounds for the development of sprouts and seedlings respectively. Sucrose levels rise when hexoses decrease apparently terminating cell division in initiating differentiation and storage activities.
Early ear development relies upon concurrent photosynthate, as the developing seed cannot utilize stored photoassimilates present in other plant tissues. Because the seed are weak sinks, it is unable to attract stored reserves from source tissues. Seed abortion may occur when concurrent photosynthate is insufficient to meet the needs of reproductive growth, resulting in dramatically decreased yield, or in the case of maize ear, barreness. The ability to manipulate source sink interactions to enhance sink strength of the ear and immature seed would make these reserves accessible, maintain seed growth, and as a consequence, buffer these important and vulnerable periods of yield formation during ear and early kernel development.
Anthesis is generally recognized as the critical period of ear and kernel development in maize. Varied experimental approaches demonstrate that treatments, which decrease the plant carbon exchange rate (CER) around anthesis, decrease grain yield. For example, large yield losses occur when maize plants are shaded (Early et al., 1967; Schussler and Westgate, 1991; Andrade et al., 1993), defoliated (Tollenaar and Daynard, 1978), subjected to water-deficits (Denmead and Shaw, 1960; Claassen and Shaw, 1970; Moss and Downey, 1971; Westgate and Boyer, 1986; Schussler and Westgate, 1991) or exposed to high plant density (Prine, 1971; Baenziger and Glover, 1980) around anthesis. Conversely, treatments that increase plant CER around anthesis increase grain yield. For example, yield enhancements are obtained when maize plants are provided supplemental radiation (Schoper et al., 1982; Ottman and Welch, 1988). In all cases, the variation in yield was directly related to the number of kernels that developed and supply of concurrent photosynthate. Collectively, these results suggest that kernel number may be limited by carbohydrate supply, particularly during drought stress at anthesis. According to the invention, enhancing sink strength of the immature ear and grain would make these limited assimilate supplies more accessible, maintain ear and seed growth, and as a consequence buffer this important vulnerable period of yield formation.
Traditional methods of improving yield formation have centered around breeding techniques. As with any valuable plant species, breeders have long used conventional breeding techniques to improve yield. While improvements have been achieved, breeding techniques are laborious and slow because of the time required to breed and grow successive plant generations. Furthermore, certain phenotypes may be impossible to obtain by conventional techniques. Thus, it would be desirable to utilize recombinant DNA technology to produce new plant varieties and cultivars in a controlled and predictable manner. It would be especially desirable to produce crop and ornamental plants with improved seed set over a range of environmental conditions to increase yield potential.
The partitioning of sucrose and starch is regulated by enzymes. Invertases are regarded as a control element in the changing carbohydrate status of seeds. Two enzymes are involved in catalyzing the cleavage process of sucrose, sucrose synthase and invertase. It has been proposed that each operates in a pathway of specific significance. In general, in sink tissues the invertase pathway is directed towards growth and cell expansion, whereas the sucrose synthase pathway is associated with storage product biosynthesis. Sucrose cleavage catalyzed by cell wall bound invertase occurs in the placento-chalazal cells of developing maize kernels. It has been implicated as a necessary step in either carbohydrate transport out of the vascular system or into the endosperm. Invertase activity seems to be important to early seed growth. The invertase pathway therefore appears to be associated with cell division and growth rather than storage.
Seed tissues actively engaged in storage often have a markedly low level of acid invertase activity but high levels of sucrose synthase activity. Sucrose synthase has frequently been cited as a marker for sink strength and the onset of starch synthesis is accompanied by an increase in enzyme activity.
In developing corn kernels a similar relationship between soluble acid invertase and import was evident during the very earliest phases of development. Both were associated with rapid rises in levels of mRNAs for soluble invertases which would be expected to peak prior to maximum accumulation in activity of the encoded enzyme. Sucrose synthase often predominates in starch/sucrose storage sinks while acid invertase predominates where cell expansion is active.
Starch synthesis takes place in the plastids of plant cells and involves ADP-Glucose Pyrophosphorylase (AGPase) which converts G1P into ADP-Glucose the direct precursor starch. AGPase is thought to control the starch-biosynthetic pathway in a number of plant species. Starch consists of two components. Linear helical amylose and branched amylopectin, both of which are glucose polymers. Amylose is composed of alpha-1,4-glucans synthesized by granule bound starch synthase isoform 1 (GBSS1) which transfers the glucosyl residue from ADP-Glucose to alpha-1,4-glucans. The combined action of soluble starch synthases and branching enzymes result in the production of amylopectin that contains additional alpha-1,6-glycocytic branch plates.
Transgenic methods for affecting starch and sugar metabolism have been tried in plants. For example, transgenic tobacco plants over-expressing a yeast-derived invertase, one of several plant enzymes involved in sucrose metabolism, showed stunted internodal elongation, reduced leaf growth, and a disturbed sink-source relationship (See, e.g., Sonnewald, U., et al. (1991) Plant J. 1:95-106). Some of these deleterious effects could be obviated by using a chemically inducible plant gene expression system (Caddick, M. X., et al. (1998) Nature Biotechnology, 16:177-180).
In tomato, suppression or over-expression of acid invertase modified the sucrose content of fruit (Fitzmaurice, C. L., et al. (1991) International patent application number PCT/US92/01385; Bennett, A. B. and Klann, E. M. (1994) U.S. Pat. No. 5,658,773).
Constitutive expression of a yeast invertase in all cells of transgenic tobacco and potato plants modified the distribution of assimilates to effect changes in habit and yield (Willmitzer, L., et al., (1993) U.S. Pat. No. 5,658,773).
Antisense inhibition of AGPase reduced starch levels in transgenic potato tubers compared to wild-type (Müller-Röber, B., et al. (1992) EMBO J. 11:1229-1238).
Over-expression of an E. coli AGPase regulatory mutant in transgenic tobacco calli, tomato leaves, and potato tubers results in increased starch production (Kishore, G. M. (1991) International Patent application number PCT/US91/04036; Stark, D. M., et al. (1992) Science, 258:287-292).
Antisense inhibition of AGPase reduced starch and increased sucrose levels in pea (Gurgess, D. G. and Dooner, H. K. (1993) U.S. Pat. No. 5,498,831). Although these studies indicate importance of sucrose and starch metabolism, there remains a need in the art for a reliable transgenic method of increasing yield stability in plants.
It can be seen from the foregoing that a need exists in the art for a transgenic method of increasing yield potential in crop and ornamental plants.
It is an object of the present invention to provide expression constructs which when expressed in a temporal and spatial manner in a transgenic plant increase yield potential, as well as resistance to stress through regulation of sink strength.
It is yet another object of this invention to provide transgenic plant lines with heritable phenotypes which are useful in breeding programs designed to increase yield potential in crop plants over a range of environmental conditions.
It is yet another object of this invention to produce seed which will produce plants with increased yield potential.
It is yet another object of this invention to provide plants, plant cells, and plant tissues containing the expression constructs of the invention.
Other objects of the invention will become apparent from the description of the invention which follows.